br Some ancillary cytological methods have already been
Some ancillary cytological methods have already been tested in clinical studies which identified additional patients with malignancy over routine AEB071 without additional false positives. These include mutational analysis (ie, KRAS, p16, p53) , immunohistochemistry (ie, mesothelin) , DNA ploidity tests . All of these tests have shown promise in accurately identifying malignant pancreatobiliary strictures, but with the exception of FISH, none of them are used in clinical practice at the present. The detection of polysomy by fluorescence in situ hybridization (FISH), has been shown to double sensitivity in the most comprehensive study to date , more recently the same group developed a set of FISH probes that separate pancreatobiliary malignancy with a sensitivity of 64.7% . The evaluation of brushings by both routine cytology and FISH is now common day practice in some cytopathology laboratories .
In recent years there has been a dramatic increase in the discovery of microRNAs (miRs) playing important roles in a variety of fundamental cellular processes and helping the early diagnosis of various diseases, mainly
cancers . MicroRNAs are disease specific, stable markers that can be detected quickly and reproducibly by PCR based methodology available more widely in regular cytopathology laboratories . Aims of the project were: (1) to prove that microRNAs can be detected and isolated from brush cytology samples, (2) to determine the expression of four tumor-associated microRNAs (miR-16, miR-21, miR-221 and miR-196a) on obtained cytology samples, (3) to give way to novel single or combined molecular markers in order to increase the sensitivity of brush cytology enough to impact clinical decision making.
PATIENTS/MATERIALS AND METHODS
Subjects and study design. Patients were prospectively enrolled at the Department of Interventional Gastroenterology, National Institute of Oncology, Budapest, Hungary. A total of 73 samples from Caucasian patients were included into this study. Samples were collected during n=57 ERCP procedures from pancreatobiliary strictures. Patients were categorized in the malignant group with (1) histological proof of malignancy (endoscopic or percutaneous biopsy, surgical exploration and sampling, autopsy), (2) clearly malignant clinical course over at least 12 months after sampling (evidence of progression such as large vessel involvement, appearance of malignant lymph nodes or new metastases on imaging), (3) pancreatobiliary tumor-related death during follow-up. Patients grouped under benign stricture had none of the mentioned features and were followed-up for 20 months to exclude progression or malignancy.
After exclusion of duplicates (repetitive sampling for suspected false negatives), parapapillary lesions, metastatic biliary strictures from non-pancreatobiliary primaries, and samples where metastatic origin of a pancreatic mass could not be ruled out, we ended up with n=35 samples (biliary malignant n=14, pancreatic malignant n=12, pancreatobiliary benign n=9). For a summary of patient characteristics we refer to Table 1.
The study protocol was approved by the Institutional Review Board and the Hungarian National Ethical Review Committee ETT-TUKEB (2372/2012/EKU). All patients gave written informed consent for sample collection and epigenetic analysis before the onset of endoscopy. Sample and data management was done anonymously.
Sample procurement. Brush cytology samples were collected during ERCP procedures using a through-the-scope disposable gastrointestinal cytology brush (Ref# 152, ConMed, Utica, NY, USA). After preparation and fixation of direct smears for routine cytology analysis during the procedure, the distal brush tip containing the